Tumor Necrosis Factor (TNF) is a pro-inflammatory cytokine that is involved in a variety of biological activities, through its binding to two distinct cell surface receptors, TNFR-1 and TNFR-2 (Tartaglia and Goeddel, Immunol. Today 13: 151-153, 1992; Rothe, et al., Immunol. Rev. 11: 81-90, 1992; Baker and Reddy, Oncogene 17: 3261-3270, 1998). Both TNF receptors are part of the larger TNF receptor superfamily (Smith, et al., Cell 76: 959-62, 1994; Grell, J Inflamm. 47: 8-17, 1995), which includes CD27, CD30, CD40 and Fas antigen, among others. These receptors share no obvious sequence similarities in the cytoplasmic domain, with the exception of TNFR-1 and Fas, which each have an ˜80 amino acid ‘death domain’ (DD) at the C-terminal with ˜28% sequence identity. These death domains can induce apoptosis by mediating self association of both TNFR-1 and Fas upon ligand binding to each receptor, a critical event to trigger downstream signaling pathways by recruiting and activating receptor-associated effector molecules (Boldin, et al., J Biol Chem 270: 7795-7798, 1995; Tartaglia, et al., Immunol. Today 13: 151-153, 1992). Recently, many of these downstream signaling proteins were identified and shown to contain a DD, which mediates the interaction with the receptor through a DD-DD interaction. For example, the DD of TRADD (TNFR-1 associated death domain protein) (Hsu, et al., Cell 81: 495-504, 1995), and MADD (Schievella, et al, J Biol Chem 272: 12069-75, 1997) have been shown to interact with TNFR1; FADD (Boldin, et al., J Biol Chem 270: 7795-7798, 1995; Chinnaiyan, et al., Cell 81: 505-512, 1995) and RIP (Stanger, et al., Cell 81: 513-523, 1995) have been shown to interact with FAS.
TRADD, one of the earlier TNFR-1 adapter proteins identified (Hsu, et al., Cell 81: 495-504, 1995), is a 34 kD protein that is recruited to the TNFR1 in a TNF dependent manner. TRADD contains two functionally separate domains, which allow the protein to couple to at least two distinct signaling pathways (Hsu, et al, Cell 84: 299-308, 1996). The C-terminal region of the protein (aa196-301) contains a death domain that mediates the interaction between TRADD and the death domains of TNFR1, FADD and RIP. The recruitment of FADD initiates the activation of the caspase cascade, which eventually leads to apoptosis. The N-terminal region of TRADD (N-TRADD) spanning from residues 1-169 appears to be a novel domain since a BLAST (Altschul, et al., Nucl. Acids. Res. 25: 3389-3402, 1997) search did not identify any sequence homology to known proteins. N-TRADD is responsible for the binding of TRAF2, a TNFR-associated factor (Hsu, et al, Cell 84: 299-308, 1996). This interaction is mediated through the TRAF domain located in the C-terminal region of TRAF2 (348-501), termed C-TRAF2. The interaction of N-TRADD with C-TRAF2 initiates TRAF2 mediated signaling processes central to the cellular inflammatory response, such as JNK and NF-kB activation (Reinhard, et al., EMBO J. 16: 1080-1092, 1997; Song, et al., Proc. Natl. Acad. Sci. 94: 9792-9796, 1997; Rothe, et al., Immunol. Rev. 11: 81-90, 1995; Cao, et al., Nature 383: 443-446, 1996). This crucial role of N-TRADD in TNF signaling is supported by the observation that the expression of N-TRADD (aa1-194) can inhibit TNF-mediated NF-κB and JNK activation in a dominant negative manner (Kieser, et al., EMBO J. 18: 2511-2521, 1999).
In addition to the TNFR-1 pathway, TRADD is also involved in LMP1 (Epstein-Barr virus latent membrane protein 1) mediated pathogenesis. LMP1 is a transforming viral oncogene product that recruits both TRADD and TRAF2 to exert its biological activities in the cell, which include activation of NF-κB, JNK and AP1 Juan, Curr. Opin Cell Biol. 9: 247-251, 1997; Farrell, Trends Microbio. 3: 105-109, 1998). Two domains in the C-terminus of LMP1 initiate the signaling processes. The CTAR1 domain binds to TRAF2, and the CTAR2 domain binds to TRADD. Although there are similarities between TNFR-1 and LMP1 in their adapter proteins, the signaling mechanisms differ. In LMP1 it is the N-TRADD region (Kieser, et al., EMBO J. 18: 2511-2521, 1999) that interacts with LMP1, instead of the DD region as it occurs with TNFR-1 (Hsu, Cell 81: 495-504, 1995). Also, whereas a dominant negative mutant of TRADD (1-194) can block both NF-κB and JNK signaling in the TNFR-1 pathway, only NF-κB activity is blocked by N-TRADD (1-194) in the LMP-1 signaling pathway (Kieser, et al., EMBO J. 18: 2511-2521, 1999).
Recently, structures of the DD of Fas (Huang, et al., Nature 384: 638-41, 1996), p75 (Liepinsh, et al., EMBO J. 16: 4999-5005, 1997) and FADD (Jeong, et al., J Biol Chem 274: 16337-42, 1999) have been solved, providing insight into the mechanisms by which they regulate apoptosis. In order to understand how N-TRADD may interact with the adapter protein TRAF2, the inventors have determined the three dimensional structure of N-TRADD (1-169) by NMR spectroscopy. The solution structure of N-TRADD consists of 5 alpha helices and four beta strands, arranged in a unique fashion. Using the structure, together with site-directed mutagenesis, a region of N-TRADD has been identified that interacts with C-TRAF2. This information, in addition to the recently published structures of C-TRAF2 (Park, et al., Nature 398: 533-538, 1999; PDB Accession Nos. 1CA4 and 1CA9; McWhirter, et al., Proc. Natl. Acad. Sci. USA 96: 8408-8413, 1999; PDB Accession No. 1QSC), provides insight into the interaction of N-TRADD and C-TRAF2, which is critical for the design and selection of potent and selective inhibitors of TNF signaling pathways.